Staphylococcal enterotoxins (SE) are unusual bacterial exotoxins in that they have the ability to cause two distinct human illnesses, food poisoning and toxic shock syndrome (TSS). SE belong to a family of pyrogenic toxins (PT) produced by Staphylococcus aureus and Streptococcus pyogenes. The PT family, which contains prototypic microbial superantigens, shares a set of immunomodulating biological properties that appear to contribute to the pathogenesis of TSS. SE association with good poisoning results from a separate ability to induce emesis when ingested. We formed several hypotheses to analyze the structural and functional organization of PT using the Type C SE (SEC) group as a model. The first three aims will determine residues necessary for emesis or T cell receptor and Class II MHC binding by mutation of SEC1 nd SEC3, subtypes on which we have the most detailed structure-function data. Information made available by our solution of the SEC3 crystal structure has allowed us to prioritize regions of SEC molecules and provided a logical approach to focus our investigation. A unique aspect of this proposal is our ability to address whether altered biological activity is due to changes in conformation rather than modification of critical residues. This will be accomplished through crystallographic analysis in addition to conventional methods. Since the SEC3 structure has already been solved, refinement to higher resolution (Aim 5) and structural determination of mutants can be done in a timely manner. As part of the mapping of functional molecular regions, we will also identify immunogenic regions on the three classical SEC subtypes, in addition to five novel SEC variants that we recently identified, as well as their ability to induce a protective immune response. Information on the topology of functional regions will contribute to understanding the molecular and immunological mechanisms by which these toxins mediate disease. Potential practical applications such as toxoid and immunotherapeutic agent development requires a dissection of critical toxic and immunogenic regions.